Purified proteolytic enzyme and method of purification

ABSTRACT

A purified protease is prepared by adjusting the pH of a protease solution to a value of between 6 and 9 and by keeping the solution at this pH and at 20-35° C. for at least 15 min and at most 120 min, so as to use the proteolytic activity of the protease to destroy the lipolytic activity of the lipases and phospholipases in the reaction medium and the pH of the solution is reduced to a value of less than or equal to 3.5.  
     The purified protease allows in particular the manufacture of infant formulae containing lecithin which are stable during storage, that is to say which do not exhibit significant degradation of the added lecithin.

[0001] The invention relates to a purified proteolytic enzyme and to amethod of purifying a proteolytic enzyme, in particular trypsin.

[0002] Commercial proteases, in particular commercial trypsin, evenafter purification by a special treatment, for example by doublecrystallization, contains residual lipases, in particular phospholipaseA₂, which is particularly resistant to the heat deactivation to whichprotease is subjected after its use in a hydrolysis process.

[0003] Trypsin is commonly used in the manufacture of proteinhydrolysates intended in particular to enter into the composition ofinfant products. To incorporate the protein portion into a finishedproduct, for example an infant milk, any residual lipolytic enzymaticactivity resulting from the protein hydrolysate must be removed. This isnecessary in order to avoid the appearance of products of degradation oflecithin which is added to the final formula for technological reasons,for example to enhance the wettability of powders, into lysolecithin, inparticular during storage. Such breakdown products may manifestthemselves both in liquid products and in powders by the appearance ofstability or organoleptic defects, for example spots, poor taste, or bytheir toxicity leading to side effects, for example of an inflammatorytype in breastfeeding infants.

[0004] However, it is the case that the complete removal ofphospholipases in particular is difficult to achieve. The completepurification of proteases generally requires various precipitationsteps, chromatographic separations, heat treatments under well-definedconditions or chemical inactivations. The complete removal ofphospholipase A₂, which is very heat-resistant, requires a prolongedheat treatment which unfortunately also affects the protease.

[0005] The aim of the invention is the preparation of a purifiedprotease whose proteolytic activity is quantitatively and qualitativelypreserved, but which is free of lipolytic activity, in particular ofphospholipase A₂, by a simple and inexpensive method.

[0006] A method of preparing purified trypsin, described for example inU.S. Pat. No. 3,886,043, is known in which a buffer solution ofcrystallized trypsin is chromatographed by passing over a resinconsisting of a dextran gel with grafted sulphonic groups, with the aimof separating the various active forms of porcine trypsin.

[0007] It is also known to prepare a lipase-free microbial rennet, forexample by the method described in U.S. Pat. No. 4,136,201, by culturingMucor miehei on an appropriate nutrient medium.

[0008] The invention relates to a purified proteolytic enzymaticpreparation, characterized in that it possesses a residual phospholipaseA₂ activity of at most 20 mU/g of pure enzyme detectable by highperformance chromatography analysis of phospholipids after incubatingwith an infant formula whose phospholipase A₂ activity is notdetectable, and in that its protease activity is maintained at not lessthan 75% of the initial activity of the enzyme.

[0009] The measurements of the enzymatic activities are detailed in theexamples hereinafter. In particular, “nondetectable” is understood tomean a residual phospholipase A₂ activity <6 mU/g of enzyme.

[0010] The enzyme may be any protease of plant, microbial or animalorigin or of biogenetic origin. It is preferably a protease of animalorigin, such as pancreatin, particularly trypsin of porcine origin.

[0011] The method according to the invention is characterized in that:

[0012] 1) the pH of a solution of the protease is adjusted to a value ofbetween 6 and 9 and in that the solution is kept at this pH and at20-35° C. for at least 15 min and at most 120 min, so as to use theproteolytic activity of the protease to destroy the lipolytic activityof the lipases and of the phospholipases of the reaction medium, and

[0013] 2) the pH of the solution is reduced to a value of less than orequal to 3.5, it being possible to reverse the order of steps 1) and 2)above.

[0014] In a preferred embodiment which makes it possible to also removetraces of residual lipases other than phospholipase A₂, the methodcomprises a final heat treatment step, preferably by UHT. Traces ofheat-sensitive lipases are thus removed.

[0015] Preferably, the adjustment of the pH to the alkaline region takesplace before the reduction of the pH to the acidic region, since it isthus possible to defer the use of the protease. In the variant where thetwo steps are reversed, the protease should be used immediately afterthe treatment.

[0016] In a preferred embodiment, there is added to the reaction mediuma magnesium salt which is soluble in the latter, preferably at thebeginning of the reaction, which makes it possible to stabilize theproteases while promoting the degradation of the phospholipases.Magnesium chloride is preferably added in an amount of 10 to 200 mM/l ofthe reaction medium, for example 50 to 100 mM/l of reaction medium.

[0017] The pure protease concentration in the solution before treatmentmay be between 0.5 and 6%, and is preferably about 2.5% by weight. Theinvention also relates to a method of preparing an infant formula basedon protein hydrolysate, characterized in that a whey product isenzymatically hydrolysed by means of a purified protease above, in thatthe hydrolysate is treated at 75-85° C./3-5 min, in that liquid fat andminerals are added thereto, in that a UHT treatment is carried out at125-135° C./2-3 min, then in that carbohydrates, vitamins and traceelements are added thereto, in that the liquid product is sterilized byUHT and in that it is aseptically packaged.

[0018] According to a variant of this method, the liquid is dried, inparticular spray-dried, after UHT sterilization treatment.

[0019] The purified enzyme according to the invention may be usedoutside the food area in the applications of proteases, for example inthe preparation of a nutritional, cosmetic or pharmaceuticalcomposition.

[0020] There may be mentioned, in this regard, anti-inflammatoryapplications, the treatment of digestive disorders, the treatment ofthromboses, the treatment of injuries and wounds and the elimination ofnecrosed tissues for example.

[0021] The examples below illustrate the invention. In these examples,the parts and percentages are by weight, unless otherwise stated.

EXAMPLE 1

[0022] 1 kg of commercially available porcine trypsin 6.0 S (Novo,Denmark) is dissolved in 10 kg of demineralized water at 25° C., withstirring, in a vessel, the protease concentration being 9.1% and theinitial pH 5. To ensure that undissolved protease is not carried over tothe next step, the solution is transferred to a new vessel.

[0023] A dilute aqueous NaOH solution at 1 M/l is added to adjust the pHof the solution to 8. The pH is then kept constant for 15 min byaddition, as required, of the aqueous NaOH solution above, for exampleby means of a pH-stat, with stirring, so as to hydrolyse thephospholipases.

[0024] After this treatment, the proteases are stabilized, that is tosay trypsin and chymotrypsin, by reducing the pH of the reaction mediumto 3 by addition of an aqueous HCl solution at 1 M/l. The solution ofproteases may be used immediately in a hydrolysis reaction or stored,for example, at −25° C. for a deferred use.

[0025] The analyses below show the enzymatic activity of the purifiedproteolytic enzyme obtained according to the invention compared withthat of the original commercially available crystallized enzyme (trypsinPTN 6.0 S, Novo, Denmark) which has not been subjected to the treatmentaccording to the invention.

[0026] 1. Determination of phospholipases:

[0027] 1.1 Determination of phospholipase A₂ by a radioisotope method.

[0028] The method is based on the cleavage of phosphatidylcholine(C14-dioleyl) by phospholipase A₂ and the punctual radiometric detectionof the labelled fractions after chromatographic separation.

[0029] 1.2 Determination of the total phospholipases by titrimetry.

[0030] The method is not specific to phospholipase A₂ and detects allphospholioases. It is based on the titration of fatty acids releasedfrom egg yolk phospholipids (Fluka, Buchs, Switzerland) byphospholipases at pH 8 (maintained by a pH-stat) and at a constanttemperature of 40° C. with 1.4 mM of sodium deoxycholate and 3 mM CaCl₂.2 g of purified egg yolk phospholipid are used with addition of 250 mgof turkey egg white trypsin inhibitor (Sigma, St. Louis, USA) per 1 g oftrypsin.

[0031] 2. Determination of lipase and esterase:

[0032] 2.1 The activity of the lipases is determined by titrimetry usingolive oil as substrate in an amount of 100 g/l at a constant pH of 8.9(pH-stat) in the presence of 1.25 g/l of taurocholate and 82.5 g/l ofgum arabic.

[0033] 2.2 The activity of the esterases is determined using the abovemethod (2.1) but taking medium-chain triglycerides (MCT) as substrate.

[0034] 3. Determination of proteases:

[0035] 3.1. For trypsin, the method described by Erlanger et al. inArch. Biochem. Biophys. 95, 271-278 is used.

[0036] 3.2. For chymotrypsin, the US Pharmacopoeia XXI (1985) method isused.

[0037] The results of the analyses of activities in the enzymepreparation are indicated in Table 1 below: TABLE 1 Phos- Tryp- Chymo-Enzyme Phospho- pholi- Lip- Ester- sin trypsin prepara- lipase A₂ pasesases ase (g/ (USP/ tion (U/g), 1.1 (U/g) (U/g) (U/g) kg) mg) Purified<0.0022 0.79 0.21 0.22 223 41 trypsin PTN 6.0 S according to theinvention Original 87 14 0.34 0.39 213 42 trypsin PTN 6.0 S

[0038] It is observed that the treatment makes it possible toconsiderably reduce the activity of enzymes other than proteases, inparticular to remove that of phospholipase A₂, while maintaining intactthe proteolytic activity in terms of quality and quantity, in particularthe equilibrium between trypsin (93% activity relative to the untreatedenzyme) and chymotrypsin (86% activity relative to the untreatedenzyme).

EXAMPLE 2

[0039] 1 kg of commercially available porcine trypsin 6.0 S (Novo,Denmark) is dissolved in 10 kg of demineralized water at 25° C., withstirring, in a vessel, the protease concentration being 9.1% and theinitial pH 5. To ensure that undissolved protease is not carried over tothe next step, the solution is transferred to a new vessel.

[0040] 224 g of magnesium chloride (MgCl₂.6 H₂O) are added, the pH isadjusted to 8.5 over 15 min with a dilute aqueous NaOH solution at 1M/l. The medium is allowed to react or 120 min at 25° C. withoutcontrolling the pH, so as to hydrolyse the phospholipases.

[0041] After this treatment, the proteases, that is to say trypsin andchymotrypsin, are stabilized by reducing the pH of the reaction mediumto 3 by addition of an aqueous HCl solution at 1 M/1 and the solution isallowed to stand for 16 h at 4° C. The purified trypsin solution is thenready for use.

[0042] It is observed that the relative activity of the trypsin is 93%of that of the original trypsin and that the relative activity of thechymotrysin is 86% of that of the original chymotrypsin.

EXAMPLE 3

[0043] The purified enzymatic preparation of Example 2 is used toprepare a hypoallergenic infant formula. Whey proteins are hydrolysedand then the hydrolysate is treated at 75-85° C./3-5 min, fat andminerals are added thereto, a UHT treatment is carried out at 125° C./2min, then maltodextrin and vitamins are added, the liquid product issterilized by UHT at 148° C./5 s and aseptically packaged.

[0044] To test the residual enzymatic activity, 10 ml of purifiedtrypsin solution according to Example 1 are added to 100 ml of theliquid infant formula above whose phospholipase A₂ activity is <6 mU/gtrypsin. After mixing, the activities of the lipase and esterase arereduced by a heat treatment at 75-80° C./3-5 min on a water bath. Aftercooling to room temperature, 55 mg of sodium azide are added and thesolution is incubated at 40° C./4 d. After incubation, the phospholipidcomposition is analysed by high-performance liquid chromatography (HPLC)and the phospholipase A₂ activity calculated, expressed in mU/100 g ofproduct (mU -PL-A₂), from the differences in the concentration of thelysophospholipids according to the formula:

mU PL−A₂=(LPC+LPE) at time T2 minus (LPC+LPE) at time t1 in mg/100 g ofproduct 540 (molecular mass of egg lysolecithins)×(t1−t2)

[0045] with LPC=lysophosphatidylcholine andLPE=lysophosphatidylethanolamine,

[0046] as well as this value expressed in terms of the concentration ofpure trypsin g/100 g, that is to say mU PL-A₂/g of pure trypsin.

[0047] The degradation of trypsin and or chymotrypsin is also evaluatedin % relative to the initial activity, as well as the degradation of thephospholipids after 9 months of storage at 20° C. in % of the originalphospholipids.

[0048] The results are indicated in Table 2 below: TABLE 2 Degrada-PL-A₂ tion of (mU/g of Chymo- the phos- Liquid pure Trypsin trypsinpholipids infant trypsin), (% of (% of (% of formula HPLC initial)initial) initial) Hydro- 16 94 75 <1 lyzed by purified trypsin PTN 6.0 Saccording to the invention Hydro- 349000 100 100 100 lyzed by theoriginal trypsin PTN 6.0 S

EXAMPLE 4

[0049] An infant formula is prepared as in Example 3, except that aphospholipid is added to the liquid mixture before spray-drying it. Itobserved that the activity of the phospholipases is strongly linked tothe degradation of the phospholipids in the product. Large load volumes,an interrupted production, associated with a high phospholipase activitydegrades the phospholipids to a certain degree before the product isdried. The products containing the purified trypsin according to theinvention show a considerably lower degradation of the addedphospholipids, depending on the total quantity of phospholipids added tothe formula, <1%, whereas it represents 20 to 90% when the same trypsinis used which has not been subjected to the purification treatmentaccording to the invention.

[0050] Furthermore, SDS-PAGE analysis of the residual proteins andanalysis of the immunologic ally active antigens by ELISA did not showsignificant differences using purified trypsin according to theinvention compared with production with the unpurified trypsin.

1. Purified proteolytic enzymatic preparation, characterized in that itpossesses a residual phospholipase A₂ activity of at most 20 mU/g ofpure enzyme detectable by high performance chromatography analysis ofphospholipids after incubating with an infant formula whosephospholipase A₂ activity is not detectable, and in that its proteaseactivity is maintained at not less than 75% of the initial activity ofthe enzyme.
 2. Preparation according to claim 1, characterized in thatthe enzyme is porcine trypsin.
 3. Method of purifying a proteolyticenzyme, in particular trypsin, characterized in that: 1) the pH of asolution of the protease is adjusted to a value of between 6 and 9 andin that the solution is kept at this pH and at 20-35° C. for at least 15min and at most 120 min so as to use the proteolytic activity of theprotease to destroy the lipolytic activity of the lipases and of thephospholipases of the reaction medium, and 2) the pH of the solution isreduced to a value of less than or equal to 3.5, it being possible toreverse the order of steps 1) and 2) above.
 4. Method according to claim3, characterized in that it comprises a final heat treatment step,preferably by UHT, which makes it possible to also remove traces ofresidual lipases other than phospholipase A₂.
 5. Method according toclaim 3, characterized in that the adjustment of the pH between 6 and 9takes place before the reduction of the pH to a value of less than orequal to 3.5.
 6. Method according to claim 3, characterized in thatthere is added to the reaction medium a magnesium salt which is solublein the latter, preferably at the beginning of the reactor, which makesit possible to stabilize the proteases while promoting the degradationof the phospholipases.
 7. Method according to claim 6, characterized inthat magnesium chloride is added in an amount of 10 to 200 mM/l andpreferably in an amount of 50 to 100 mM/l of reaction medium.
 8. Methodaccording to claim 3, characterized in that the pure proteaseconcentration in the solution before treatment is between 0.5 and 6%,and is in particular about 2.5% by weight.
 9. Method of preparing aninfant formula based on protein hydrolysate, characterized in that awhey product is enzymatically hydrolysed by means of a purified proteaseaccording to claim 1, in that the hydrolysate is treated at 75-85°C./3-5 min, in that liquid fat and minerals are added thereto, in that aUHT treatment is carried out at 125-135° C./2-3 min, then in thatcarbohydrates, vitamins and trace elements are added thereto, in thatthe liquid product is sterilized by UHT and in that it is asepticallypackaged.
 10. Method according to claim 9, characterized in that afterthe UHT sterilization treatment, the liquid is dried, in particularspray-dried.
 11. Use of a purified enzymatic preparation according toclaim 1, in the preparation of a nutritional, cosmetic or pharmaceuticalcomposition.